Targeted CAR T-Cell Integration for Uniform TCR-Free Expression
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Solution Overview
Problem
Current genetic modification techniques for T cells, such as those using chimeric antigen receptors (CARs), suffer from heterogeneous expression, insertional mutagenesis, and potential autoimmunity or graft versus host disease due to random integration and TCR expression, limiting their effectiveness and safety in cancer and autoimmune disorder treatments.
Innovation Solution
Integrating a transgene under the control of an endogenous promoter within the T cell genome, using targeted homologous recombination, to achieve precise and controlled expression of therapeutic proteins or nucleic acids, reducing variability and safety risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If random integration methods (viral vectors or transposons) are used to deliver CAR genes, then T cell modification is achieved, but heterogeneous CAR expression and insertional mutagenesis occur
Solution Approach 1:
The patent uses a specific genomic locus (CCR5 or TRAC) as an intermediary integration site to achieve controlled transgene insertion. This mediator approach replaces random integration with targeted integration at a predetermined safe harbor locus, ensuring uniform CAR expression while avoiding insertional mutagenesis of other genes
Solution Approach 2:
The patent changes the integration parameter from random to targeted by using CRISPR/Cas9 or other genome editing tools to direct integration to specific loci. This parameter change ensures that the transgene integrates at a controlled location with appropriate chromatin environment for consistent expression
2Adaptability or versatility
If T cells express TCR along with CAR, then antigen recognition capability is maintained, but autoimmunity and graft versus host disease risk increase
Solution Approach 1:
The patent extracts or removes the TCR from the T cell system by integrating the CAR construct into the TRAC locus, which replaces the endogenous TCR alpha chain gene. This extraction eliminates TCR-mediated antigen recognition that causes autoimmunity and GvHD while preserving CAR-mediated target cell recognition through the engineered receptor
3Manufacturing precision
If CCR5 locus is used for transgene integration, then targeted delivery is achieved, but susceptibility to West Nile virus infection increases
Solution Approach 1:
The patent changes the integration locus parameter from CCR5 to TRAC (T cell receptor alpha chain) to avoid the harmful effect of increased West Nile virus susceptibility associated with CCR5 disruption, while maintaining the benefits of targeted integration for uniform transgene expression
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach ensures consistent and optimal T cell function, minimizing insertional mutagenesis and alloreactivity, enhancing the therapeutic efficacy of T cells for cancer and autoimmune disorders.
Implementation Method 1
integrating the transgene at a specific site within the genome of the T cell using a homologous recombination system
Data Source
AI summary
The invention provides a T cell wherein one or more therapeutic transgenes is integrated at a within the genome of the cell such that expression of the transgene is under control of an endogenous promoter of the T cell. The invention additional provides methods of making and using such cells to treat a subject with T cell therapy. The invention also provides a T cell wherein a recombinant nucleic acid sequence encoding a chimeric antigen receptor (CAR) is integrated at a first site within the genome of the cell such that the CAR is expressed by the cell at the surface of the cell, and wherein integration of the nucleic acid encoding the CAR at the first site reduces or prevents expression of a functional T cell receptor (TCR) complex at the surface of the cell. The invention additional provides methods of making and using such cells to treat a subject with CAR therapy.